Tlr7 agonist maleate salt, crystalline forms c, d and e thereof, preparation methods and uses of maleate salt and crystalline forms

ABSTRACT

The present invention relates to a maleate salt of a compound represented by formula I, a method for preparing the salt, a pharmaceutical composition containing the salt, and the use of the salt. The present invention also relates to crystalline forms C, D and E of the maleate salt of the compound represented by formula I, methods for preparing the crystalline forms, crystalline compositions and pharmaceutical compositions containing the crystalline forms, and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase of International Application No.PCT/CN2017/072890, filed on Feb. 4, 2017, which claims priority toChinese Patent Application No. 201610082030.0, filed on Feb. 5, 2016,each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the field of medicinal chemistry, andparticularly relates to a maleate of TLR7 agonist, a preparing processthereof, a pharmaceutical composition comprising the same and usethereof. The present invention also relates to crystal form C, crystalform D and crystal form E of the salt, preparing processes, crystallinecompositions comprising the crystal forms, pharmaceutical compositionscomprising the crystal forms or crystalline compositions and usethereof.

BACKGROUND

Toll-like receptor is expressed by various immune cells and recognizeshigh reserved structural motifs: Pathogen Associated Molecular Pattern(PAMP) expressed by microorganism pathogens or Damage AssociatedMolecular Patterns (DAMP) released by dead cells. PAMP or DAMPstimulates Toll-like receptor to trigger signal cascade which inducesthe activations of transcriptional factors like AP-1, NF-κB andinterferon regulators (pulse response function). It results in variouscell responses, including productions of interferons, proinflammatorycytokines and effector cytokines, whereby immune response is produced.By far, 13 types of Toll-like receptors have been discovered in mammal.Toll-like receptors 1, 2, 4, 5 and 6 are mainly expressed on the cellsurface while Toll-like receptors 3, 7, 8 and 9 are expressed in theendosome. Different Toll-like receptors recognize ligands derived fromdifferent pathogens. Toll-like receptor 7 (TLR7) is mainly expressed byplasmaeytoid dendritic cells (pDC), and recognized via ligand to inducethe secretion of interferon α (IFN-α). Toll-like receptor 7 (TLR7) andToll-like receptor 8 (TLR8) are highly homologous and therefore theligand of TLR7 in most cases is also that of TLR8. TLR8 stimulationmainly induces the productions of cytokines like tumor necrosis factor α(TNF-α) and chemoattractant. Interferon a is one of the medicines fortreating chronic hepatitis B or hepatitis C while TNF-a is aproinflammatory cytokine, of which the over secretion will result severeside effects.

There have been reported several TLR7 agonists, like imiquimod (BritishJournal of Dermatology 2003; 149 (Suppl. 66): 5-8), resiquimod(Antiviral Research 64 (2004) 79-83), GS-9620 (Gastroenterology (2013),144(7), 1508-1517). Nevertheless, it is desirable to have novel TLR7agonists with better selectivity, activity and safety.

Chinese Patent Application No. 201410405136.0 which is incorporated inits entirety by reference herein discloses one small molecule, i.e.2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amineand preparing process thereof, which has the following structure:

SUMMARY

In an aspect, provided is a maleate of the compound of formula I:

In another aspect, provided are crystal forms of the maleate of thecompound of formula I, the corresponding preparing process and thecrystalline composition. In one embodiment, the crystal form is crystalform C, crystal form D or crystal form E.

In one embodiment, the crystal form C is characterized by an X-raypowder diffraction (XRPD) pattern having diffraction peaks at2θ=7.6°±0.2°, 9.9°±0.2°, 17.8°±0.2°, 22.8°±0.2°, 24.2°±0.2°, 26.3°±0.2°.

In one embodiment, the crystal form D is characterized by an X-raypowder diffraction (XRPD) pattern having diffraction peaks at2θ=5.1°±0.2°, 9.5°±0.2°, 11.2°±0.2°, 17.6°±0.2°, 20.2°±0.2°, 23.0°±0.2°.

In one embodiment, the crystal form E is characterized by an X-raypowder diffraction (XRPD) pattern having diffraction peaks at2θ=4.9°±0.2°, 5.3°±0.2°, 9.0°±0.2°, 16.5°±0.2°, 19.3°±0.2°.

In another aspect, provided is a pharmaceutical composition, comprisingone or more crystal forms or the crystalline composition thereofaccording to the invention. The pharmaceutical composition can furtheroptionally comprise pharmaceutically acceptable carrier, excipientand/or medium.

In another aspect, provided is a method for treating or preventingToll-like receptor 7 (TLR7) associated disease, comprising administeringto a subject in need thereof the crystal form or the crystallinecomposition or the pharmaceutical composition according to the inventionin an effective amount. Preferably, the disease is virus infection.

In a further aspect, provided is use of the crystal form or thecrystalline composition or the pharmaceutical composition according tothe invention for the manufacture of a medicament for treating orpreventing Toll-like receptor 7 (TLR7) associated disease. Preferably,the disease is virus infection.

In a yet further aspect, provided is the crystal form or the crystallinecomposition or the pharmaceutical composition according to the inventionfor use in treating or preventing Toll-like receptor 7 (TLR7) associateddisease. Preferably, the disease is virus infection.

In one embodiment of the invention, the virus infection is hepatitisvirus infection, particularly hepatitis B or hepatitis C virusinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: XRPD pattern of the crystal form C of the maleate of thecompound of formula I.

FIG. 2: XRPD pattern of the crystal form D of the maleate of thecompound of formula I.

FIG. 3: XRPD pattern of the crystal form E of the maleate of thecompound of formula I.

DETAILED DESCRIPTION

General Definition and Terminology

Unless stated otherwise, the terms and phrases used herein have thefollowing meaning. A specific term or phrase shall not be considered asunclear or indefinite when it is not specifically defined. It should beunderstood according to the general meaning. The trade name used hereinrefers to the corresponding product or the active ingredient.

Unless specifically defined otherwise, proportion (including percentage)or part is calculated based on weight herein.

When used with a numerical variable, the term “approximate” or “about”usually refers to the value of the variable and all the values of thevariable within the experimental error (for example, within an average95% confidence interval) or within ±10% of the specified value, or awider range.

The expression “comprise” or its synonyms “contain”, “include”, “have”or the like is open-ended, which does not exclude other unlistedelements, steps or ingredients. The expression “consist of” excludes anyunlisted elements, steps or ingredients. The expression “substantiallyconsist of ” refers to specified elements, steps or ingredients within agiven range, together with optional elements, steps or components whichdo not substantively affect the basic and novel feature of the claimedsubject matter. It should be understood that the expression “comprise”encompasses the expressions “substantially consist of” and “consist of”.

The term “optional” or “optionally” means the event described subsequentthereto may or may not happen. This term encompasses the cases that theevent may or may not happen.

The term “pharmaceutical composition” refers to an active ingredient,which is optionally combined with one or more pharmaceuticallyacceptable components (for example, but not limited to carrier and/orexcipient). The active ingredient is exemplified as the compound offormula I or maleate thereof, one or more crystal forms according to theinvention, or one or more crystalline compositions according to theinvention.

The term “pharmaceutically acceptable carrier” refers to those carrierswhich have no significant irritation and do not impair the bioactivityand property of the active compound. The “pharmaceutically acceptablecarrier” refers to inert substance which is administered with activeingredient and is beneficial to the administration thereof, andcomprises but not limited to any of the following substances approved byState Food and Drug Administration for use in human or animal (e.g.livestock): glidant, sweetening agent, diluent, preservative,dye/colorant, flavoring agent, surfactant, wetting agent, dispersant,disintegrant, suspending agent, stabilizing agent, isotonic agent,solvent or emulsifying agent. Non-limiting examples of the carrierscomprise calcium carbonate, calcium phosphate, various sugars andstarches, cellulose derivative, gelatine, vegetable oil and polyethyleneglycol or the like. Other information regarding the carriers may befound in Remington: The Science and Practice of Pharmacy, 21st Ed.,Lippincott, Williams & Wilkins (2005), of which the contents areincorporated herein by reference. The term “excipient” generally refersto the carrier, diluent and/or medium used to formulate effectivepharmaceutical composition.

The term “administration” or “administrating” or the like refers to amethod that enables a compound or composition to be delivered to adesired site of biological action. Such methods comprise but not limitedto oral, parenteral (including intravenous, subcutaneous,intraperitoneal, intramuscular, intravascular injection or infusion),local, rectal administration or the like.

As for pharmaceutical or pharmacological active agent, the term“effective amount” refers to the amount of the medicament or agent whichis not toxic but sufficient to achieve the desired effect. With respectto the oral formulation herein, the “effective amount” for an activesubstance in the composition refers to the amount required to achievethe desired effect in combination with another active substance in thecomposition. The effective amount may be determined individually anddepends on the age and general condition of the receptor as well asspecific active substance. The effective amount in specific case can bedetermined by a person skilled in the art through conventional test.

The term “active ingredient”, “therapeutic agent”, “active substance” or“active agent” refers to a chemical entity useful for treating orpreventing target disorder, disease or condition effectively. The termherein may refer to for example the compound of formula I or maleatethereof, one or more crystal forms according to the invention or one ormore crystalline compositions according to the invention.

In X-ray powder diffraction (XRPD or XRD) spectra, the diffractionpattern obtained from crystalline compound is generally characteristicfor a particular crystal form in which the relative intensities of thebands (especially at low angles) may vary with the dominant orientationeffect due to the difference of crystallization conditions, particlediameters, and other measuring conditions. Therefore, the relativeintensities of diffraction peaks are not characteristic for the givencrystal form. It is more important to note the relative positions ofpeaks rather than their relative intensities when determining whetherthe crystal form is the same as that known in the art. In addition,there may be slight errors in the positions of the peaks for any givencrystal form, which is also well known in the art of crystallography.For example, the position of the peak may shift due to the change intemperature, sample movement or instrument calibration during analysisof the sample; and the measuring error of 2θ value may sometimes beabout ±0.2°, typically about ±0.1°. Therefore, this error should betaken into account when determining the crystal structure. If thecrystal forms according to the invention are described as substantiallyas shown in the figure, the term “substantially” is also intended toencompass such differences in the diffraction peak.

In the XRPD pattern, the peak position is usually represented by angle2θ or crystal surface distance d and a simple conversion between d and θis d=λ/2sin θ, where d represents the crystal surface distance, λrepresents the wavelength of the incident X-ray, and θ is diffractionangle. As for the same crystal form of the same compound, the peakpositions of the XRPD pattern are similar as a whole, and the relativeintensity error may be large. It should also be noted that, inidentification of a mixture, some diffraction lines may be lost due tothe factors like decrease in content, etc., and thus it is not necessaryto rely on the entire bands observed in the high purity sample, and evenone band may be characteristic for a given crystal.

Differential scanning calorimetry (DSC) is used to measure thetransition temperature when a crystal absorbs or releases heat due tochanges in its crystal structure or crystal melting. The thermaltransition temperature and the melting point error are typically withinabout 5° C., usually about 3° C. for the same crystal form of the samecompound in a continuous analysis. When a compound is described ashaving a given DSC peak or melting point, it means the DSC peak ormelting point ±5° C. Provided is an auxiliary method by DSC to identifydifferent crystal forms. Different crystal forms may be identifiedaccording to their different transition temperature characteristics. Itis noted that the DSC peak or melting point of the mixture may vary overa wide range. In addition, the melting temperature is associated withthe rate of temperature rise due to the decomposition during the meltingof the substance.

Differential scanning calorimetry (DSC) herein is measured by thefollowing method: apparatus: TA Q2000 differential scanning calorimeter;method: a sample (˜1 mg) is placed in a DSC aluminum pan, method: 25°C.˜300° C., heating rate 10° C./min.

The term “crystalline composition” refers to a solid form, whichcomprises one or more crystal forms according to the invention (such as,crystal form C, D and/or E). The amounts of crystal forms contained inthe crystalline composition may be 50% or more, 80% or more, 90% ormore, or 95% or more independently. In addition to crystal formaccording to the invention, the crystalline composition may alsooptionally comprise other crystal or amorphous form of the compound offormula I or the salt thereof (such as maleate) or the impurities otherthan these substances. It should be understood by those skilled in theart that the sum of contents of the components in the crystallinecomposition should be 100%.

Maleate of the Compound of Formula I

Provided is a maleate of the compound of formula I:

In a preferred embodiment, in maleate of the compound of formula I, themolar ratio of the compound of formula Ito maleic acid is 1:1˜3,preferably 1:2. The maleate of the compound of formula I can be preparedby conventional maleate preparing process.

Crystal Form C

Provided is crystal form C of maleate of the compound of formula I,which has diffraction peaks at 2θ=7.6°±0.2°, 9.9°±0.2°, 17.8°±0.2°,22.8°±0.2°, 24.2°±0.2°, 26.3°±0.2° in X-ray diffraction (XRPD) pattern.

In a specific embodiment, the crystal form C has diffraction peaks at2θ=5.6°±0.2°, 7.6°±0.2°, 9.9°±0.2°, 17.8°±0.2°, 19.8°±0.2°, 22.8°±0.2°,24.2°±0.2°, 25.0°±0.2°, 26.3°±0.2° in X-ray diffraction (XRPD) pattern.

In a more specific embodiment, the crystal form C has diffraction peaksat 2θ=5.6°±0.2°, 6.0°±0.2°, 7.6°±0.2°, 9.9°±0.2°, 12.0°±0.2°,15.3°±0.2°, 17.8°±0.2°, 18.5°±0.2°, 19.8°±0.2°, 20.4°±0.2°, 22.8°±0.2°,23.1°±0.2°, 24.2°±0.2°, 24.7°±0.2°, 25.0°±0.2°, 26.3°±0.2° in X-raydiffraction (XRPD) pattern.

In a particular embodiment, diffraction peaks of the crystal form C ofthe maleate of the compound of formula I are characterized as follows:

Number 2θ ± 0.2(°) Relative intensity (%) 1 5.6 22.0 2 6.0 18.7 3 7.6100.0 4 9.4 8.0 5 9.9 87.7 6 10.2 9.8 7 11.1 13.8 8 12.0 16.5 9 12.9 2.510 15.3 18.7 11 16.6 5.5 12 16.9 7.0 13 17.8 30.0 14 18.1 9.4 15 18.515.5 16 19.1 9.9 17 19.8 25.4 18 20.4 18.1 19 20.7 14.1 20 21.3 5.8 2121.7 5.6 22 22.4 5.1 23 22.8 41.9 24 23.1 17.7 25 24.2 42.1 26 24.7 19.727 25.0 26.6 28 25.4 3.7 29 25.9 8.7 30 26.3 35.2 31 28.0 8.3 32 28.610.9 33 29.3 4.4 34 30.4 7.2 35 33.2 3.5

In an embodiment, the X-ray powder diffraction pattern of the crystalform C of the maleate of the compound of formula I is substantiallyshown in FIG. 1.

The crystal form C can also be characterized by DSC, with initialtemperature of 97.0° C.±5° C. and peak temperature of 106.0° C.±5° C.

In an embodiment, in crystal form C, the molar ratio of the compound offormula Ito maleic acid is 1:1-3, preferably 1:2.

Provided is also a process for preparing the crystal form C accordingthe invention, comprising precipitating the maleate of the compound offormula I from a solvent.

In an embodiment, the process comprises the following steps:

-   1) dissolving the compound of formula I in a solvent, which is    preferably heated to promote dissolution;-   2) adding maleic acid; and-   3) cooling for crystallization, filtering, washing and drying to    obtain the crystal form C.

In step 1), the solvent is selected from the group consisting ofmethanol, ethanol, propanol, isopropanol, n-butanol, isobutanol,tertiary butanol, acetone, ethyl acetate and mixed solvent thereof;preferably ethanol.

In step 1), the amount of solvent added per 1 mol of the compound offormula I is 0.2 to 8L, preferably 0.3 to 4L, more preferably 0.5 to 2L,most preferably 1L.

In step 1), the heating temperature may be 40° C. to 90° C., preferably50° C. to 80° C., more preferably 70° C. to 80° C.

In step 2), the amount of maleic acid added per 1 mol of the compound offormula I is 1.0 to 4.0 mol, preferably 1.6 to 3.0 mol, more preferably2.0 to 2.4 mol, most preferably 2.2 mol.

Provided is also a crystalline composition comprising the crystal formC. In an embodiment, based on the weight of the crystalline composition,the content of crystal form C is 50% or more, preferably 80% or more,more preferably 90% or more, and most preferably 95% or more. Thecrystalline composition, in addition to the crystal form C, can alsocomprise the compound of formula I or salt thereof in other crystal oramorphous forms, or impurities other than these substances.

Crystal Form D

Provided is crystal form D of the maleate of the compound of formula I,which has diffraction peaks at 2θ=5.1°±0.2°, 9.5°±0.2°, 11.2°±0.2,17.6±0.2°, 20.2°±0.2°, 23.0°±0.2 ° in X-ray diffraction (XRPD) pattern.

In a specific embodiment, the crystal form D has diffraction peaks at2θ=5.1°±0.2°, 9.5°±0.2°, 11.2°±0.2°, 17.6°±0.2°, 20.2°±0.2°, 20.7°±0.2°,23.0°±0.2°, 23.7°±0.2° in X-ray diffraction (XRPD) pattern.

In a more specific embodiment, the crystal form D has diffraction peakat 2θ=5.1°±0.2°, 5.6°±0.2°, 9.5°±0.2°, 11.2°±0.2°, 16.9°±0.2°,17.6°±0.2°, 20.2°±0.2°, 20.7°±0.2°, 22.6°±0.2°, 23.0°±0.2°, 23.7°±0.2°,24.5°±0.2° in X-ray diffraction (XRPD) pattern.

In a particular embodiment, the diffraction peaks of the crystal form Dof the maleate of the compound of formula I are characterized asfollows:

Number 2θ ± 0.2(°) Relative intensity (%) 1 5.1 100.0 2 5.6 11.5 3 7.02.7 4 8.7 2.6 5 9.5 36.2 6 10.3 3.1 7 11.2 23.5 8 12.0 3.1 9 14.2 2.5 1016.2 4.2 11 16.9 11.2 12 17.6 33.6 13 18.3 4.7 14 18.8 2.0 15 19.2 5.316 19.6 4.4 17 20.2 35.6 18 20.7 19.0 19 21.4 3.5 20 22.6 12.3 21 23.020.1 22 23.7 17.7 23 24.5 14.8 24 26.4 6.1 25 27.1 3.1 26 27.7 3.3 2728.4 5.2 28 28.8 8.7 29 30.7 3.8

In an embodiment, the X-ray powder diffraction pattern of the crystalform D of the maleate of the compound of formula I is substantiallyshown in FIG. 2.

The crystal form D can also be characterized by DSC, with initialtemperature of 98.3° C.±5° C. and peak temperature of 110.1° C.±5° C.

In an embodiment, in crystal form D the molar ratio of the compound offormula Ito maleic acid is 1:1-3, preferably 1:2.

Provided is also a process for preparing the crystal form D, comprisingthe following steps:

-   1) placing the crystal form C of the maleate of the compound of    formula I in acetone solvent to form a suspension;-   2) shaking at constant temperature;-   3) centrifugating, washing and drying to obtain the crystal form D.

In step 1), the amount of acetone added per 1 g of crystal form C of themaleate of the compound of formula I is 2 to 30 mL, preferably 8 to 24mL, more preferably 12 to 20 mL, most preferably 14 to 16 mL.

In step 2), the constant temperature is 20° C. to 60° C., preferably 30°C. to 50° C., more preferably 35° C. to 45° C., most preferably 40° C.

Provided is also a crystalline composition comprising the crystal formD. In an embodiment, based on the weight of the crystalline composition,the crystal form D is 50% or more, preferably 80% or more, morepreferably 90% or more, and most preferably 95% or more. Furthermore,the crystalline composition, in addition to the crystal form D, can alsocomprise compound of formula I or salt thereof in other crystal oramorphous forms, or impurities other than these substances.

Crystal Form E

Provided is crystal form E of the maleate of the compound of formula I,which has diffraction peaks at 2θ=4.9°±0.2°, 5.3°±0.2°, 9.0°±0.2°,16.5°±0.2°, 19.3°±0.2° in X-ray diffraction (XRPD) pattern.

In a specific embodiment, the crystal form E has diffraction peaks at2θ=4.9°±0.2°, 5.3°±0.2, 6.7°±0.2°, 9.0°±0.2°, 10.8°±0.2°, 16.5°±0.2°,19.3°±0.2 ° in X-ray diffraction (XRPD) pattern.

In a more specific embodiment, the crystal form E has diffraction peaksat 2θ=4.9°±0.2°, 5.3°±0.2°, 6.7°±0.2°, 9.0°±0.2°, 10.8°±0.2°,16.2°±0.2°, 16.5°±0.2°, 19.3°±0.2°, 22.0°±0.2°, 22.6°±0.2°, 25.9°±0.2°in X-ray diffraction (XRPD) pattern.

In a specific embodiment, the diffraction peaks of the crystal form E ofthe maleate of the compound of formula I are characterized as follows:

Number 2θ ± 0.2(°) Relative intensity (%) 1 4.9 100.0 2 5.3 10.4 3 6.76.9 4 8.2 2.8 5 8.6 2.7 6 9.0 7.9 7 10.8 5.1 8 16.2 3.9 9 16.5 15.4 1017.7 2.0 11 19.3 7.2 12 22.0 4.6 13 22.6 4.0 14 24.3 2.3 15 25.9 3.4 1627.1 2.8

In an embodiment, the X-ray powder diffraction pattern of the crystalform E of the maleate of the compound of formula I is substantiallyshown in FIG. 3.

The crystal form E can also be characterized by DSC with initialtemperature of 85.7° C.±5° C. and peak temperature of 97.5° C.±5° C.

In an embodiment, in crystal form E, the molar ratio of the compound offormula Ito maleic acid is 1:1-3, preferably 1:2.

Provided is also a process for preparing the crystal form E, comprisingthe following steps:

-   1) placing the crystal form C of the maleate of the compound of    formula I in isopropanol solvent to form a suspension;-   2) shaking at constant temperature;-   3) centrifugating, washing and drying to obtain the crystal form E.

In step 1), the amount of isopropanol added per 1 g of the crystal formC of the maleate of the compound of formula I is 2 to 30 mL, preferably8 to 24 mL, more preferably 12 to 20 mL, further preferably 14 to 16 mL.

In step 2), the constant temperature is 20° C. to 60° C., preferably 30°C. to 50° C., more preferably 35° C. to 45° C., further preferably 40°C.

Provided is also a crystalline composition comprising the crystal formE. In an embodiment, based on the weight of the crystalline composition,crystal form E is 50% or more, preferably 80% or more, more preferably90% or more, and most preferably 95% or more. The crystallinecomposition, in addition to crystal form E, can also comprise thecompound of formula I or salt thereof in other crystal or amorphousforms, or impurities other than these substances.

Pharmaceutical Composition and Administration

Provided is a pharmaceutical composition, which comprises maleate of thecompound of formula I; the crystal form C or the crystalline compositioncomprising the crystal form C; the crystal form D or the crystallinecomposition comprising the crystal form D; or the crystal form E or thecrystalline composition comprising the crystal form E, or anycombination thereof in an effective amount. Furthermore, thepharmaceutical composition also may or may not comprise pharmaceuticallyacceptable carrier, excipient and/or medium.

The compound according to the invention is administrated in a pure formor in a suitable pharmaceutical composition form, which can be performedvia any acceptable administration mode of the agent with similar use.Pharmaceutical composition according to the invention may be prepared bycombining of the compound according to the invention or the salt thereofwith a suitable pharmaceutically acceptable carrier, for example it maybe formulated into solid, semi-solid, liquid or gas formulation, such astablet, pill, capsule, powder, granule, ointment, emulsion, suspension,solution, suppository, injection, inhalant, gel, microsphere, aerosol orthe like.

The pharmaceutical composition according to the invention may beprepared by the processes well-known in the art, such as conventionalmixing, dissolution, granulation, dragee coating, levigation, emulsion,freeze-drying or the like.

Typical routes for administering the compound according to the inventionor the pharmaceutical composition thereof comprise but not limited tooral, rectal, transmucosal, enteral administration or local,transcutaneous, inhalant, parenteral, sublingual, intravaginal,intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous,intravenous administration.

In a preferred embodiment, the pharmaceutical composition is in an oraladministration form. As for oral administration, the active compoundsmay be mixed with the pharmaceutically acceptable carriers, excipientsand/or media well-known in the art to prepare the pharmaceuticalcomposition. The carriers, excipients and media may be used to preparethe compounds according to the invention into tablet, pill, troche,dragee, capsule, liquid, gel, slurry, suspension or the like useful fororal administration to the patient.

Solid oral composition may be prepared by conventional mixing, fillingor compressing processes, for example, by the following processes:mixing the active compound with solid excipients, optionally milling theresultant mixture, adding other proper adjuvants if necessary, and thenprocessing the mixture into granules so as to obtain the core of tabletor dragee.The proper excipients comprise but not limited to filler, suchas sugar, including lactose, sucrose, mannitol or sorbitol; cellulosepreparation such as microcrystalline cellulose, maize starch, wheatstarch, rice starch and potato starch; and other substances, such assilica gel, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxymethyl cellulose sodium and/or polyvinylpyrrolidone;disintegrant, such as carboxymethyl starch sodium, croscarmellosesodium, crosslinked polyvinylpyrrolidone, agar or alginic acid. Saltsuch as sodium alginate may also be used. The core of the dragee may beoptionally coated through well-known processes in general pharmaceuticalpractice, especially by enteric coating.

Beneficial Effects

The crystal form C, crystal form D and crystal form E of the maleate ofthe compound of formula I according to the invention have the advantagesof high purity, high crystallinity and good stability, which aresuitable for the manufacture of a medicament for preventing or treatingToll-like receptor 7 (TLR7) associated disease.

The technical solutions of the invention are illustrated according tothe following paragraphs [1] to [44]:

-   [1] A maleate of the compound of formula I,

-   [2] The maleate of the compound of formula I according to paragraph    [1], characterized in that, the molar ratio of the compound of    formula Ito the maleic acid is 1:1-3, preferably 1:2.-   [3] A pharmaceutical composition, comprising the maleate of the    compound of formula I according to paragraph [1] or [2] in an    effective amount.-   [4] Use of the maleate of the compound of formula I according to    paragraph [1] or [2] or the pharmaceutical composition according to    paragraph [3] for the manufacture of a medicament for treating    toll-like receptor 7 associated disease.-   [5] The use according to paragraph [4], characterized in that, the    disease is virus infection, particularly hepatitis virus infection,    for example hepatitis B or hepatitis C virus infection.-   [6] A crystal form C of the maleate of the compound of formula I,    characterized in that it has diffraction peaks at 2θ=7.6°±0.2°,    9.9°±0.2°, 17.8°±0.2°, 22.8°±0.2°, 24.2°±0.2°, 26.3°±0.2° in X-ray    powder diffraction pattern.-   [7] The crystal form C according to paragraph [6], characterized in    that it has diffraction peaks at 2θ=5.6°±0.2°, 7.6°±0.2°, 9.9°±0.2°,    17.8°±0.2°, 19.8°±0.2°, 22.8°±0.2°, 24.2°±0.2°, 25.0°±0.2°,    26.3°±0.2° in X-ray powder diffraction pattern.-   [8] The crystal form C according to paragraph [7], characterized in    that it has diffraction peaks at 2θ=5.6°±0.2°, 6.0°±0.2°, 7.6°±0.2°,    9.9°±0.2°, 12.0°±0.2°, 15.3°±0.2°, 17.8°±0.2°, 18.5°±0.2°,    19.8°±0.2°, 20.4°±0.2°, 22.8°±0.2°, 23.1°±0.2°, 24.2°±0.2°,    24.7°±0.2°, 25.0°±0.2°, 26.3°±0.2° in X-ray powder diffraction    pattern.-   [9] The crystal form C according to any one of paragraphs [6]-[8],    characterized in that, it has X-ray powder diffraction pattern    substantially shown in FIG. 1.

[10] The crystal form C according to any one of paragraphs [6]-[9],characterized in that, when characterized by DSC, the initialtemperature is 97.0° C.±5° C. and the peak temperature is 106.0° C.±5°C.

-   [11] A process for preparing the crystal form C according to any one    of paragraphs [6]-[10], comprising the following steps:-   1) dissolving the compound of formula I in a solvent, which is    preferably heated to promote dissolution;-   2) adding maleic acid;-   3) cooling for crystallization, filtering, washing and drying to    obtain the crystal form C.-   [12] The preparing process according to paragraph [11],    characterized in that the solvent in step 1) is selected from the    group consisting of methanol, ethanol, propanol, isopropanol,    n-butanol, isobutanol, tertiary butanol, acetone, ethyl acetate and    mixed solvent thereof.-   [13] The preparing process according to paragraph [12],    characterized in that the solvent is ethanol.-   [14] The preparing process according to any one of paragraphs    [11]-[13], characterized in that, in step 1), the amount of solvent    added per 1 mol of the compound of formula I is 0.2 to 8 L,    preferably 0.3 to 4 L, more preferably 0.5 to 2 L, most preferably 1    L.-   [15] The preparing process according to any one of paragraphs    [11]-[14], characterized in that, in step 2), the amount of maleic    acid added per 1 mol of the compound of formula I is 1.0 to 4.0 mol,    preferably 1.6 to 3.0 mol, more preferably 2.0 to 2.4 mol, most    preferably 2.2 mol.-   [16] The preparation process according to any one of paragraphs    [11]-[14], characterized in that, in step 1), the heating    temperature may be 40° C. to 90° C., preferably 50° C. to 80° C.,    more preferably 70° C. to 80° C.-   [17] A crystalline composition, characterized in that, based on the    weight of the crystalline composition, the crystal form C according    to any one of paragraphs [6]-[10] is 50% or more, preferably 80% or    more, more preferably 90% or more, and most preferably 95% or more.

[18] A pharmaceutical composition, comprising the crystal form Caccording to any one of paragraphs [6]-[10] or the crystallinecomposition according to paragraph [17] in an effective amount.

[19] Use of the crystal form C according to any one of paragraphs[6]-[10] or the crystalline composition according to paragraph [17] orthe pharmaceutical composition according to paragraph [18] for themanufacture of a medicament for treating Toll-like receptor 7 (TLR7)associated disease.

[20] The use according to paragraph [19], characterized in that thedisease is virus infection, particularly hepatitis virus infection, suchas hepatitis B or hepatitis C virus infection.

[21] A crystal form D of the maleate of the compound of formula I,characterized in that it has diffraction peaks at 2θ=5.1°±0.2°,9.5°±0.2°, 11.2°±0.2°, 17.6°±0.2°, 20.2°±0.2°, 23.0°±0.2° in X-raypowder diffraction pattern.

-   [22] The crystal form D according to paragraph [21] characterized in    that it has diffraction peaks at 2θ=5.1°±0.2°, 9.5°±0.2°,    11.2°±0.2°, 17.6°±0.2°, 20.2°±0.2°, 20.7°±0.2°, 23.0°±0.2°,    23.7°±0.2° in X-ray powder diffraction pattern.-   [23] The crystal form D according to paragraph [22], characterized    in that it has diffraction peaks at 2θ=5.1°±0.2°, 5.6°±0.2°,    9.5°±0.2°, 11.2°±0.2°, 16.9°±0.2°, 17.6°±0.2°, 20.2°±0.2°,    20.7°±0.2°, 22.6°±0.2°, 23.0°±0.2°, 23.7°±0.2°, 24.5°±0.2° in X-ray    powder diffraction pattern.-   [24] The crystal form D according to any one of paragraphs    [21]-[23], characterized in that, the crystal form D has X-ray    powder diffraction pattern substantially shown in FIG. 2.-   [25] The crystal form D according to any one of paragraphs    [21]-[24], characterized in that, when characterized by DSC, the    initial temperature is 98.3° C. ±5° C. and the peak temperature is    110.1° C. ±5° C.-   [26] A process for preparing the crystal form D according to    paragraph [25], comprising the following steps:-   1) placing the crystal form C of the maleate of the compound of    formula I in acetone solvent to form a suspension;-   2) shaking at constant temperature;-   3) centrifugating, washing and drying to obtain the crystal form D.-   [27] The preparing process according to any one of paragraphs    [21]-[26], characterized in that, in step 1), the amount of acetone    added per 1 g of crystal form C of the maleate of the compound of    formula I is 2 to 30 mL, preferably 8 to 24 mL, more preferably 12    to 20 mL, most preferably 14 to 16 mL.-   [28] The preparing process according to any one of paragraphs    [21]-[27], characterized in that, in step 2), the constant    temperature is 20° C. to 60° C., preferably 30° C. to 50° C., more    preferably 35° C. to 45° C., most preferably 40° C.-   [29] A crystalline composition, characterized in that, based on the    weight of the crystalline composition, the crystal form D according    to any one of paragraphs [21]-[25] is 50% or more, preferably 80% or    more, more preferably 90% or more, and most preferably 95% or more.-   [30] A pharmaceutical composition, comprising the crystal form D    according to any one of paragraphs [21]-[25] or the crystalline    composition according to paragraph [29] in an effective amount.-   [31] Use of the crystal form D according to any one of paragraphs    [21]-[25] or the crystalline composition according to paragraph [29]    or the pharmaceutical composition according to paragraph [30] for    the manufacture of a medicament for treating Toll-like receptor 7    (TLR7) associated disease.-   [32] The use according to paragraph [31], characterized in that the    disease is virus infection, particularly hepatitis virus infection,    such as hepatitis B or hepatitis C virus infection.-   [33] A crystal form E of the maleate of the compound of formula I    characterized in that it has diffraction peaks at 20=4.9°±0.2°,    5.3°±0.2°, 9.0°±0.2°, 16.5°±0.2°, 19.3°±0.2° in X-ray powder    diffraction pattern.-   [34] The crystal form E according to paragraph [33] characterized in    that it has diffraction peaks at 20=4.9°±0.2°, 5.3°±0.2°, 6.7°±0.2°,    9.0°±0.2°, 10.8°±0.2°, 16.5°±0.2°, 19.3°±0.2° in X-ray powder    diffraction pattern.-   [35] The crystal form E according to paragraph [34], characterized    in that it has diffraction peaks at 2θ=4.9°±0.2°, 5.3°±0.2°,    6.7°±0.2°, 9.0°±0.2°, 10.8°±0.2°, 16.2°±0.2°, 16.5°±0.2°,    19.3°±0.2°, 22.0°±0.2°, 22.6°±0.2°, 25.9°±0.2° in X-ray powder    diffraction pattern.-   [36] The crystal form E according to any one of paragraphs    [33]-[35], characterized in that, the crystal form E has X-ray    powder diffraction pattern substantially shown in FIG. 3.-   [37] The crystal form E according to any one of paragraphs    [33]-[36], characterized in that, when characterized by DSC, the    initial temperature is 85.7° C. ±5° C. and the peak temperature is    97.5° C. ±5° C.-   [38] A process for preparing the crystal form E according to any one    of paragraphs [33]-[37], comprising the following steps:-   1) placing the crystal form C of the maleate of the compound of    formula I in isopropanol solvent to form a suspension;-   2) shaking at constant temperature;-   3) centrifugating, washing and drying to obtain the crystal form E.-   [39] The preparing process according to paragraph [38],    characterized in that, in step 1), the amount of isopropanol added    per 1 g of the crystal form C of the maleate of the compound of    formula I is 2 to 30 mL, preferably 8 to 24 mL, more preferably 12    to 20 mL, further preferably 14 to 16 mL.-   [40] The preparing process according to paragraphs [38] or [39],    characterized in that, in step 2), the constant temperature is    20° C. to 60° C., preferably 30° C. to 50° C., more preferably    35° C. to 45° C., further preferably 40° C.-   [41] A crystalline composition, characterized in that, based on the    weight of the crystalline composition, the crystal form E according    to any one of paragraphs [33]-[37] is 50% or more, preferably 80% or    more, more preferably 90% or more, and most preferably 95% or more.-   [42] A pharmaceutical composition, comprising the crystal form E    according to any one of paragraphs [33]-[37] or the crystalline    composition according to paragraph [41] in an effective amount.-   [43] Use of the crystal form E according to any one of paragraphs    [33]-[37] or the crystalline composition according to paragraph [41]    or the pharmaceutical composition according to paragraph [42] for    the manufacture of a medicament for treating Toll-like receptor 7    (TLR7) associated disease.-   [44] The use according to paragraph [43], characterized in that the    disease is virus infection, particularly hepatitis virus infection,    such as hepatitis B or hepatitis C virus infection.

EXAMPLES

The following abbreviations are used herein: SEM-Cl:2-(trimethylsilyl)ethoxymethyl chloride; SEM:2-(trimethylsilyl)ethoxymethyl; DIPEA: diisopropylethylamine; TFA:trifluoroacetic acid; DMF: N,N-dimethylformamide; n-BuOH: n-butanol;NH3.H₂O: aqueous ammonia; Na: sodium; XRPD: X-ray powder diffraction;DSC: differential thermal analysis.

In the present invention, X-ray diffraction pattern was determined asfollows: apparatus: Bruker D8 ADVANCE X-ray diffractometer; method:target: Cu: K-Alpha; wavelength λ=1.54179 Å; voltage: 40 kV; current: 40mA; scanning range: 4˜40°; sample rotation speed: 15 rpm; scanningspeed: 10°/min.

The solvents used herein are commercially available and can be usedwithout further purification. The synthesis reactions in preparationexamples are generally performed under inert nitrogen atmosphere inanhydrous solvent. Data of proton magnetic resonance is recorded inBruker Avance III 400 (400 MHz) spectrometer, with the chemical shiftshown as (ppm) at tetramethylsilane low field. Mass spectrometry isdetermined on Agilent 1200 plus 6110 (&1956A). LC/MS or Shimadzu MSincludes a DAD: SPD-M20A(LC) and Shimadzu Micromass 2020 detector. Massspectrometer is equipped with an electrospray ionization (ESI) operatedat positive or negative mode.

Preparation Example 1 Preparation of2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine maleate

Formula III:

2,4-dichloro-5-((2-(trimethylsilyl)ethoxy)methyl)-5H-pyrrolo[3,2-d]pyrimidine

The compound of formula II (2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine)(4.00 kg, 21.28 mol) was dissolved in DMF (20.00 L), and DIPEA (2.58 kg,20.00 mol) was added in portions at room temperature (25° C.) followedby stirring for 30 min. The reaction liquid was cooled to 0° C. with anice bath, and then SEM-Cl (4.00 kg, 24.00 mol) was added dropwise slowlyover 5 h at a dropping rate of 1 to 2 drops per second. After addition,the reaction liquid was stirred at 0° C. for 4 h. The reaction wasmonitored by HPLC. After completion, the reaction liquid was quenchedand diluted with 70 L of water and then extracted with ethyl acetate (15L×3). The combined organic phase was washed successively with 1 Maqueous hydrochloric acid (5 L×2) and saturated brine (7 L×2), and thesolvent was removed by distillation under reduced pressure to give thecompound of formula III (6.40 kg, 20.11 mol, yield 94.50%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.24-8.35 (m, 1H), 6.70-6.85 (m, 1H), 5.77(s, 2H), 3.45-3.57 (m, 2H), 0.74-0.86 (m, 2H), 0.00 (s, 9H).

Formula IV:

2-chloro-5-((2-(trimethylsilyl)ethoxy)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

The compound of formula III (1.60 kg, 5.03 mol) was dissolved inisopropanol (1.60 L) in a 10 L clave. Aqueous ammonia (4 L) was added atroom temperature (25° C.) in one portion and the reaction mixture wasstirred at 95° C. for 7 h. The reaction was monitored by HPLC.

After completion, the reaction liquid was allowed to cool to roomtemperature and filtered through a Buchner funnel to give a dark brownsolid. The solid was successively slurried with ethyl acetate/n-heptane(1/1, 5 L×2) and ethyl acetate (4 L) to give the compound of formula IVas brown solid (1.25 kg, 4.18 mol, yield 83.1%).

¹H NMR (400 MHz, DMSO-d₆) δ 7.61-7.77 (m, 1H), 6.97-7.19 (m, 2H),6.28-6.38 (m, 1H), 5.54-5.67 (m, 2H), 3.43-3.53 (m, 2H), 0.76-0.91 (m,2H), 0.07 (s, 9H).

Formula V:

2-butoxy-5-((2-(trimethylsilyl)ethoxy)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

To n-BuOH (17.0 L) was slowly added metal sodium (525.05 g, 22.84 mol)in portions under nitrogen. After addition, the temperature of thesystem was raised to 60° C., and stirring was performed continuously atthe temperature until the metal sodium was completely dissolved. Thenthe system was cooled to 25° C., and the compound of formula IV (1.95kg, 6.53 mol) was added in portions. After being mixed homogenously withstirring, the reaction mixture was continuously stirred for 8 h at 90°C. The reaction was monitored by HPLC. After completion, the reactionmixture was allowed to cool spontaneously to 25° C., and slowly pouredinto 30 L of saturated aqueous ammonium chloride. Then the reactionmixture was extracted with ethyl acetate (15 L×3) and the combinedorganic phase was washed with saturated brine (20 L×2), dried withanhydrous Na₂SO₄, and filtered. After the solvent was distilled offunder reduced pressure, the residue was slurried in n-heptane (4 L) andthe solid was separated by filtration and slurried in ethyl acetate (5L) to give the compound of formula V as yellow-white solid (1.53 kg,4.55 mol, 69.7%).

¹H NMR (400 MHz, DMSO-d₆) δ 7.49-7.54 (m, 1H), 6.54-6.62 (m, 2H) , 6.15-6.20 (m, 1H), 5.54 (s, 2H), 4.10-4.22 (m, 2H), 3.42-3.55 (m, 2H),1.58-1.73 (m, 2H), 1.35-1.47 (m, 2H), 0.90-0.96 (m, 3H), 0.83-0.89 (m,2H), 0.05 (s, 9H).

Formula VI: 2-butoxy-5H-pyrrolo[3,2-d]pyrimidin-4-amine

The compound of formula V (1.10 kg, 3.27 mol) was dissolved in TFA (5.50L) and the reaction liquid was stirred at 25° C. for 16 h. The reactionwas monitored by HPLC. After completion, TFA was removed by distillationunder reduced pressure and the residue was dissolved in methanol (1.2 L)and ice water (1.2 L). pH of the system was adjusted to 12 withconcentrated aqueous ammonia under uniform stirring. The mixture wasstirred for 2 h and the precipitate was precipitated from the solutioncontinuously. After filtration, the filter cake as white solid wasslurried with 15% aqueous ammonia (1.2 L×3) and ethyl acetate (4 L)successively to give the compound of formula VI as white solid (550.00g, 2.67 mol, 81.7%).

¹H NMR (400 MHz, methanol-d₄) δ 7.37 (d, J=2.89 Hz, 1H), 6.29 (d, J=3.01Hz, 1H), 4.27 (t, J=6.53 Hz, 2H), 1.75 (d, J=7.91 Hz, 2H), 1.44-1.61 (m,2H), 1.00 (t, J=7.40 Hz, 3H).

Formula VII:

4-((4-amino-2-butoxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-hydroxymethyl)benzaldehyde

To a three-necked flask were added terephthalaldehyde (790.64 mg, 5.82mmol) and isopropanol (10 mL),2-butoxy-5H-pyrrolo[3,2-d]pyrimidin-4-amine (1.00 g, 4.85 mmol) wasadded with stirring, and the system was cooled to 0° C. and stirred foranother 10 min. Purified water (10 mL) and potassium carbonate (804.17mg, 5.82 mmol) were added, and reacted at 25° C. for 16 h until thereactants were depleted with the monitor by LCMS. A solid wasprecipitated out after the reaction was completed. After filtration, theresulting solid was slurried with 20 mL of purified water and 30 mL(ethyl acetate/n-heptane=1/20) successively, filtered and dried to givethe compound of formula VII as yellow solid (1.50 g, 4.41 mmol, yield:90.9%).

¹H NMR (400 MHz, methanol-d₄) δ 9.94 (s, 1H), 7.86 (d, J=8.16 Hz, 2H),7.72 (d, J=8.16 Hz, 2H), 7.12-7.17 (m, 1H), 6.19 (s, 1H), 4.28 (t,J=6.53 Hz, 2H), 1.68-1.77 (m, 2H), 1.44-1.54 (m, 2H), 0.97 (t, J=7.34Hz, 3H).

Formula VIII:

(4-amino-2-butoxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)(4-(pyrrolidin-1-ylmethyl)phenyl)methanol

To a 30 L reactor were added the compound of formula VII (450.0 g, 1.32mol) and isopropanol (4.5 L), and the mixture was stirred for 5 min.Then glacial acetic acid (119.0 g, 1.98 mol) was added and thetemperature was lowered to 0-10° C. with stirring. Pyrrolidine (112.4 g,1.58 mol) was added dropwise, with the temperature below 10° C. Afteraddition, sodium triacetoxyborohydride (420.0 g, 1.98 mol) was added inportions and reacted at 10-20° C. for 3 h until the raw materials weredepleted with the monitor by liquid chromatography. After the completionof the reaction, 5 L of purified water was added and the temperature ofthe solution was lowered to about -10° C., and 12 L of 15% aqueousammonia was added to the solution, with the solution temperature below0° C. during the addition. Solid was precipitated out under stirring.Filtration was performed and the resulting filter cake was slurried with2 L of water and 2 L×2 ethyl acetate. Filtration was performed anddrying was conducted under reduced pressure at 40° C. for 12 h to givethe compound of formula VIII as yellow solid (465.0 g, 1.18 mol, yield89.4%, moisture 0.9%).

¹H NMR (400 MHz, methanol-d₄) δ 7.46 (d, J=7.91 Hz, 1H), 7.29 (d, J=8.03Hz, 1H), 7.09 (s, 1H), 6.12 (s, 1H), 4.29 (t, J=6.53 Hz, 2H), 3.60 (s,2H), 2.52 (br. s., 4H), 1.66-1.83 (m, 6H), 1.49 (d, J=7.53 Hz, 2H), 0.98(t, J=7.40 Hz, 3H).

Formula I:

2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

To 20L clave were added the compound of formula VIII (440.0 g, 1.11mol)and dichloromethane (7.0 L), and the temperature of the system waslowered to below −15° C. with stirring. After triethylsilane (880 mL,5.55 mol) was added dropwise to the system, trifluoroacetic acid (880mL) was added dropwise continuously, with the temperature kept below−10° C. during addition. After addition, the reaction was carried out at0° C. for 2 h and monitored by liquid chromatography until the rawmaterial point disappeared. After completion of the reaction, thereaction liquid was concentrated to dryness, and 2.2 L of ethyl acetatewas added to the solution. Stirring was performed to lower thetemperature to below 0° C. Then saturated sodium carbonate solution wasadded to adjust the solution to pH 9-10, during which the systemtemperature was kept below 10° C. Filtration was performed and theresulting filter cake was slurried with 2.2 L of water. Filtration wasperformed and drying was conducted under reduced pressure to give 550 gof trifluoroacetate of the compound of formula I as white solid.

To 1.6 L of ethanol was added 525 g of trifluoroacetate of the compoundof formula I as white solid, and the temperature of the system waslowered to about 0° C. with stirring. Then 2.2 L of 1 mol/L sodiumhydroxide solution was added. Filtration was performed and the resultingfilter cake was slurried with 2.5 L of purified water. Filtration wasperformed and drying was conducted under reduced pressure to give 380.0g of the compound of formula I as solid.

¹H NMR (400 MHz, methanol-d₄) δ 7.27 (d, J=8.0 Hz, 2H), 7.22 (d, J=8.0Hz, 2H), 7.04 (s, 1H), 4.32 (t, J=6.6 Hz, 2H), 3.99 (s, 2H), 3.60 (s,2H), 2.55-2.52 (m, 4H), 1.85-1.71 (m, 6H), 1.55-1.48 (m, 2H), 1.00 (t,J=7.4 Hz, 3H).

Example 1 Preparation of Crystal Form C of Maleate of2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

To 10 mL ethanol was added the compound of formula I (3.80 g, 10 mmol),which was heated to be dissolved. Then maleic acid (2.55 g, 22 mmol) wasadded to the solution and stirring was performed with heatingcontinuously until the solution was clear. The solution was then allowedto cool to room temperature and stand for 1-2 h. The precipitate wasfiltered and the solid was dried under reduced pressure with an oil pumpto give crystal form C of maleate of the compound of formula I as solid.In the resulting maleate, the molar ratio of the compound of formula Itomaleic acid was 1: 2.

XRPD was measured as follows: apparatus: Bruker D8 ADVANCE X-raydiffractometer; method: target: Cu: K-Alpha; wavelength λ=1.54179 Å;voltage: 40 kV; current: 40 mA; scanning range: 4˜40°; sample rotationspeed: 15 rpm; scanning speed: 10°/min.

The obtained compound crystal had diffraction peaks substantially asshown in FIG. 1.

Example 2 Preparation of Crystal Form D of Maleate of2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

To 0.8 mL acetone was added crystal form C of maleate of compound offormula I (50 mg) to form a suspension. The suspension sample was shakenon a thermostat (40° C.) for 2 days (in dark). The residual solid wascentrifuged and dried in a vacuum oven at 40° C. overnight to givecrystal form D of maleate of the compound of formula I as solid. In theresulting maleate, the molar ratio of the compound of formula Ito maleicacid was 1:2.

XRPD was measured as follows: apparatus: Bruker D8 ADVANCE X-raydiffractometer; method: target: Cu: K-Alpha; wavelength λ=1.54179 Å;voltage: 40 kV; current: 40 mA; scanning range: 4˜40°; sample rotationspeed: 15 rpm; scanning speed: 10°/min.

The obtained compound crystal had diffraction peaks substantially asshown in FIG. 2.

Example 3 Preparation of Crystal Form E of Maleate of2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

To 0.8 mL isopropanol was added crystal form C of maleate of compound offormula I (50 mg) to form a suspension. The suspension sample was shakenon a thermostat (40° C.) for 2 days (in dark). The residual solid wascentrifuged and dried in a vacuum oven at 40° C. overnight to givecrystal form E of maleate of the compound of formula I as solid. In theresulting maleate, the molar ratio of the compound of formula Ito maleicacid was 1:2.

XRPD was measured as follows: apparatus: Bruker D8 ADVANCE X-raydiffractometer; method: target: Cu: K-Alpha; wavelength λ=1.54179 Å;voltage: 40 kV; current: 40 mA; scanning range: 4˜40°; sample rotationspeed: 15 rpm; scanning speed: 10°/min.

The obtained compound crystal had diffraction peaks substantially asshown in FIG. 3.

Example 4 High Temperature Stability Test

The crystal forms prepared in Examples 1-3 were tested in theaccelerated test under high temperature conditions for stability inaccordance with Guidelines for the Stability Test of PharmaceuticalIngredients and Pharmaceutical Preparations (Chinese Pharmacopoeia 2010Appendix XIXC).

The crystal forms C, D and E prepared in Examples 1-3 were placed in anopen-clean container at 60° C., respectively. The samples were taken fortest on day 10, day 20 and day 30, respectively. The results werecompared with the initial test result on day 0.

Example 5 High Humidity Stability Test

The crystal forms prepared in Examples 1-3 were tested in theaccelerated test under high humidity conditions for stability inaccordance with Guidelines for the Stability Test of PharmaceuticalIngredients and Pharmaceutical Preparations (Chinese Pharmacopoeia 2010Appendix XIXC).

The crystal forms C, D and E prepared in Examples 1-3 were subjected toan accelerated test in a constant temperature and humidity vessel underthe conditions of 40° C./75% humidity (open). The samples were taken fortest on day 30, day 60 and day 90, respectively. The results werecompared with the initial test result on day 0.

Example 6 Light Stability

The crystal forms prepared in Examples 1-3 under light conditions weretested for stability in accordance with Guidelines for the StabilityTest of Pharmaceutical Ingredients and Pharmaceutical Preparations(Chinese Pharmacopoeia 2010 Appendix XIXC).

The crystal forms C, D and E prepared in Examples 1-3 were respectivelyplaced in an illumination environment of 5000 L×±500 L×. The sampleswere taken on day 5, day 10 and day 30. The test results were comparedwith the initial test result on day 0.

Examples of Pharmacological Activity Efficacy Example 1 Toll-likereceptor 7 and Toll-like receptor 8 in vitro receptor binding activityscreen

Reagents:

-   HEK-blue hTLR7 cell and HEK-blue hTLR8 cell (available from    InvivoGen)-   DMEM medium-   heat inactivated fetal bovine serum-   Anti Mycoplasma reagent Normocin™-   bleomycin-   blasticidin

The structure of GS-9620 and R848 used are as follows, wherein thepreparation of GS-9620 could be referred to the process disclosed inUS20100143301; R848 was commercially available from ABGENT (IMG-2208,specification: 0.5 mg).

Scheme:

1. Preparation of 96-well compound plate:

The compounds were gradient diluted with DMSO in 3-fold using liquidwork station POD starting at a concentration of 10 mmol/L and 10 pointswere diluted (2nd column to 11th column, and each point was duplicated).At 12th column, 1 μL of 5 mg/mL positive compound R848 was added aspositive control; and at 1st column, 1 μL of DMSO was added as negativecontrol. Each well contained 1 μL of DMSO.

2. The cells in culture flask were collected and the cell density wasdiluted to 250,000 cells/mL.

3. 200 μL (50,000 cells/well) of cell suspension was added into preparedcompound plate and the final concentration of DMSO in each well was0.5%.

4. The culture plates containing cells and the compounds were incubatedin CO₂ incubator for 24 h at 37° C., 5%CO₂.

5. After 24 h incubation, 20 μL of supernatant was removed from eachwell to a 96-well transparent assay plate. To each well of the assayplate was added 180 μL of Quanti-Blue reagent and the plate wasincubated in an incubator at 37° C., 5%CO₂ for 1 h.

6. After 1 h, the content of alkaline phosphatase in 20 μL ofsupernatant was determined using Microplate Reader OD650.

7. EC₅₀ of each compound was obtained with Prism software.

Results are shown in Table 1:

TABLE 1 Name of the compound TLR7 EC50 (nM) TLR8 EC50 (nM) GS-9620 5177867 The compound of formula I 160 11632As is shown in table 1, the compound of formula I according to theinvention showed higher in vitro receptor binding activity to Toll-likereceptor 7 than the control (Toll-like receptor 7 agonist GS-9620) andlower in vitro receptor binding activity to Toll-like receptor 8 thanthe control (Toll-like receptor 7 agonist GS-9620).

The compound of the present invention has distinct selectivitydifferences with respect to different receptors, and the effect issuperior over the prior art.

Efficacy Example 2 Peripheral Blood Mononuclear Cell Test Scheme

The purpose of this example is to determine the expression level ofcytokines 24 h after stimulation to human peripheral blood mononuclearcells (PBMC) with the compound of formula I.

The cell supernatant was assayed without dilution and the levels ofIFN-α were directly determined. The compound of formula I was firstlyformulated into 20 mM DMSO stock solution and was gradient diluted withcell medium in 10-fold with the total number of 11 diluting points. Thecompounds in 9 diluting points (the highest concentration was 200μmol/L) were added into 96-well plate with 50 μL in each well. Freshhuman peripheral blood mononuclear cells were inoculated, with 150 μL ineach well containing 450,000 cells. The cell culture plate was incubatedin an incubator at 37° C., 5%CO₂ for 24 h. After incubation, the cultureplate was centrifuged at 1200 rpm for 5 min and the supernatant wascollected and stored at −20° C. for determination. The determination ofcytokine was performed using Cytometric Bead Array (CBA) ofBD-Pharmingen on flow cytometer. Using the above determining method, thelowest drug concentration stimulating the production of 30 pg/mL ofIFN-α was designated as the MEC value in the cytokine stimulating test.The results were shown in Table 2.

TABLE 2 Name of the compound IFN-α MEC(nM) TNF-α MEC (nM) GS-9620 50 500The compound of formula I 5 500

Conclusion: Compared with the control (GS-9620), the compound of formulaI of the invention showed better in vitro IFN-α inducing activity ofPBMCs and comparable TNF-α inducing activity.

1.-10. (canceled)
 11. A crystal form C of a maleate of a compound offormula I, wherein the crystal form C has diffraction peaks at2θ=5.6°±0.2°, 7.6°±0.2°, 9.9°±0.2°, 17.8°±0.2°, 19.8°±0.2°, 22.8°±0.2°,24.2°±0.2°, 25.0°±0.2°, 26.3°±0.2° in X-ray diffraction pattern


12. The crystal form C according to claim 11, wherein the crystal form Chas diffraction peaks at 2θ=5.6°±0.2°, 6.0°±0.2°, 7.6°±0.2°, 9.9°±0.2°,12.0°±0.2°, 15.3°±0.2°, 17.8°±0.2°, 18.5°±0.2°, 19.8°±0.2°, 20.4°±0.2°,22.8°±0.2°, 23.1°±0.2°, 24.2°±0.2°, 24.7°±0.2°, 25.0°±0.2°, 26.3°±0.2°in X-ray diffraction pattern.
 13. The crystal form C according to claim11, wherein the crystal form C has a X-ray powder diffraction patternsubstantially shown in FIG.
 1. 14. A process for preparing the crystalform C according to claim 11, comprising the following steps: 1)dissolving the compound of formula I in a solvent; 2) adding maleicacid; and 3) cooling for crystallization, filtering, washing and dryingto obtain the crystal form C, wherein the solvent is selected from thegroup consisting of methanol, ethanol, propanol, isopropanol, n-butanol,isobutanol, tertiary butanol, acetone, ethyl acetate and mixed solventthereof.
 15. The process according to claim 14, wherein the solvent isethanol.
 16. A crystal form D of a maleate of a compound of formula I,wherein the crystal form D has diffraction peaks at 2θ=5.1°±0.2°,9.5°±0.2°, 11.2°±0.2°, 17.6°±0.2°, 20.2°±0.2°, 20.7°±0.2°, 23.0°±0.2°,23.7°±0.2° in X-ray diffraction pattern


17. The crystal form D according to claim 16, wherein the crystal form Dhas diffraction peaks at 2θ=5.1°±0.2°, 5.6°±0.2°, 9.5°±0.2°, 11.2°±0.2°,16.9°±0.2°, 17.6°±0.2°, 20.2°±0.2°, 20.7°±0.2°, 22.6°±0.2°, 23.0°±0.2°,23.7°±0.2°, 24.5°±0.2° in X-ray diffraction pattern.
 18. The crystalform D according to claim 16, wherein the crystal form D has a X-raypowder diffraction pattern substantially shown in FIG.
 2. 19. A processfor preparing the crystal form D according to claim 16, comprising thefollowing steps: 1) placing a crystal form C of the maleate of thecompound of formula I in acetone solvent to form a suspension; 2)shaking at constant temperature; and 3) centrifugating, washing anddrying to obtain the crystal form D, wherein the crystal form C hasdiffraction peaks at 2θ=5.6°±0.2°, 7.6°±0.2°, 9.9°±0.2°, 17.8°±0.2°,19.8°±0.2°, 22.8°±0.2°, 24.2°±0.2°, 25.0°±0.2°, 26.3°±0.2° in X-raydiffraction pattern.
 20. A crystal form E of a maleate of a compound offormula I, wherein the crystal form E has diffraction peaks at2θ=4.9°±0.2°, 5.3°±0.2°, 6.7°±0.2°, 9.0°±0.2°, 10.8°±0.2°, 16.5°±0.2°,19.3°±0.2° in X-ray powder diffraction pattern


21. The crystal form E according to claim 20, wherein the crystal form Ehas diffraction peaks at 2θ=4.9°±0.2°, 5.3°±0.2°, 6.7°±0.2°, 9.0°±0.2°,10.8°±0.2°, 16.2°±0.2°, 16.5°±0.2°, 19.3°±0.2°, 22.0°±0.2°, 22.6°±0.2°,25.9°±0.2° in X-ray powder diffraction pattern.
 22. The crystal form Eaccording to claim 20, wherein the crystal form E has a X-ray powderdiffraction pattern substantially shown in FIG.
 3. 23. A process forpreparing the crystal form E according to claim 20, comprising thefollowing steps: 1) placing a crystal form C of the maleate of thecompound of formula I in isopropanol solvent to form a suspension; 2)shaking at constant temperature; and 3) centrifugating, washing anddrying to obtain the crystal form E, wherein the crystal form C hasdiffraction peaks at 2θ=5.6°±0.2°, 7.6°±0.2°, 9.9°±0.2°, 17.8°±0.2°,19.8°±0.2°, 22.8°±0.2°, 24.2°±0.2°, 25.0°±0.2°, 26.3°±0.2° in X-raydiffraction pattern.
 24. A pharmaceutical composition comprising thecrystal form C according to claim 11 in an effective amount.
 25. Apharmaceutical composition comprising the crystal form D according toclaim 16 in an effective amount.
 26. A pharmaceutical compositioncomprising the crystal form E according to claim 20 in an effectiveamount.
 27. A method for treating hepatitis B viral infection orhepatitis C viral infection comprising administering a subject in needthereof the crystal form C according to claim
 11. 28. A method fortreating hepatitis B viral infection or hepatitis C viral infectioncomprising administering a subject in need thereof the crystal form Daccording to claim
 16. 29. A method for treating hepatitis B viralinfection or hepatitis C viral infection comprising administering asubject in need thereof the crystal form E according to claim 20.